Epithelial tumor progression involves abnormal cell interactions with the extracellular environment as well abnormalities intrinsic to tumor cells themselves. Cutaneous squamous cell carcinoma (SCC) arises in skin from ultraviolet mutagenesis and disproportionately affects U.S. Veterans due to sun exposure incurred in military service. Nationally, SCC is the second most common cancer, with ~1 million new cases yearly. SCC can lead to disfigurement and, in rare cases, lethal metastases. This Merit Review recently identified collagens as the most highly mutated gene family in SCC, with the COL11A1 gene the second most frequently mutated gene overall in SCC (63%). It also found additional collagen genes mutated at high frequency (>20%) in SCC, including COL17A1 and COL4A4, as well as COL7A1, whose inherited mutation causes forms of epidermolysis bullosa with aggressive skin SCCs. SCC collagen mutations concentrate at prolines and glycines in the Gly-X-Y (with X and Y commonly proline) triple helical sequence, alterations known to produce dominant-negative collagens, raising the possibility that tumor-secreted mutant collagens may enable cancer in a trans-dominant fashion. Consistent with a pro-oncogenic function for mutant collagens, knocking in an SCC-associated COL11A1 mutation enhanced neoplastic invasion in vivo compared to isogenic COL11A1 wild-type control and ablating endogenously mutant COL11A1 in SCC tumors impaired tumorigenesis in vivo, indicating that mutant collagens functionally promote tumorigenesis. Aim I will test a model in which secreted mutant collagen proteins act in a non-cell autonomous fashion to accelerate tumorigenesis. First, it will determine if mutant COL11A1 can promote neoplastic progression of tumor cells in trans using mosaic tissue models. Second, it will quantify the impacts on epidermal tumor progression of mutations in multiple additional collagens that are frequently mutated in SCC, including COL17A1, COL4A4, and COL7A1. Aim I will study the action of COL11A1 in epidermal tumor progression and determine if other recurrently mutated collagens can also promote SCC. This project also recently used single-cell RNA-sequencing of 47,771 cells from a series of human SCC tumors, along with patient and site-matched normal control skin, to identify tumor cell subpopulations in SCC. This defined a new tumor-specific keratinocyte (TSK) population with no counterpart in normal tissue that expressed COL11A1 and other genes linked to cellular communication and invasion. Such tumor subpopulations enable neoplasia by communicating with each other through cell surface and secreted proteins, however, the sets of proteins ? as opposed to RNAs ? that are actually expressed in living tumors by specific tumor subpopulations have been a technical challenge to define. To address this, we developed a new proximity proteomics method, Secreted Protein Identification (SecrID) that can identify cell surface and secreted proteins within discrete cell populations of interest in heterogenous living tumor tissues in vivo to characterize cell subpopulation proteins in tumor progression. Aim II is based on a model in which the TSK SCC subpopulation uses specific cellular communication proteins to drive malignancy. First, it will use SecrID to define the secreted and cell surface proteins specific to the TSK cell subpopulation, based on the premise that these may mediate pro-neoplastic cellular communication. Second, it will ablate TSK subpopulation-specific genes, including those identified by SecrID, to define their impact on tumorigenesis and to search for new accessible therapeutic targets. Aim II will characterize the tumor-specific SCC TSK subpopulation and identify the molecules it uses to influence tumor progression. At the end of the proposed funding cycle, we plan to have characterized the actions of mutant collagens and newly defined tumor cell subpopulations in epidermal tumor progression.